基于Wnt通路探讨温阳化浊方含药血清对Ishikawa和JAr细胞胚胎种植模型的影响  

The Effect of Wenyang Huazhuo Formula(温阳化浊方)-containing Serum on Ishikawa and JAr Cell Embryo Implantation Model based on Wnt Pathway

作　　者：冯伟华 赵晓丽 陈明丽 江楠 戎蓓蕾 夏天[1] FENG Weihua;ZHAO Xiaoli;CHEN Mingli;JIANG Nan;RONG Beilei;XIA Tian(First Teaching Hospital of Tianjin University of Traditional Chinese Medicine,Tianjin,300193)

机构地区：[1]天津中医药大学第一附属医院,天津市西青区300193

出　　处：《中医杂志》2022年第20期1975-1984,共10页Journal of Traditional Chinese Medicine

基　　金：国家自然科学基金(81774351)。

摘　　要：目的 探讨温阳化浊方促进胚胎种植的可能作用机制。方法 将20只大鼠随机分为温阳化浊方组和空白组,每组10只。温阳化浊方组大鼠给予温阳化浊方药液21.05 g/kg连续灌胃7天,空白组不予灌胃,制备温阳化浊方含药血清和空白血清。体外培养THP-1单核细胞,佛波酯(PMA)诱导分化成巨噬细胞,再用20 ng/ml脂多糖(LPS)和20 ng/ml γ干扰素(IFN-γ)诱导巨噬细胞M1极化。M1型巨噬细胞分别加入2%、4%、8%、10%、15%、20%的温阳化浊方含药血清和空白血清培养,采用CCK8法检测细胞存活率,选取最大无毒剂量的温阳化浊方含药血清进行后续实验。M1型巨噬细胞分为:模型1组、温阳化浊方含药血清1组、空白血清1组,在诱导M1极化后,温阳化浊方含药血清1组给予筛选浓度的温阳化浊方含药血清,空白血清1组给予同浓度的空白血清,各组均干预24 h和48 h,Real-time PCR法检测M1型巨噬细胞标志物肿瘤坏死因子α(TNF-α)、白细胞介素1β (IL-1β)和M2型巨噬细胞标志物趋化因子配体18 (CCL-18)、甘露糖受体C型1 (MRC-1) mRNA表达。采用Transwell体系共培养M1型巨噬细胞、Ishikawa细胞和JAr细胞,建立体外胚胎种植模型。将胚胎种植模型细胞分为:非容受态组(只包含Ishikawa细胞)、容受态组(包含Ishikawa细胞和JAr细胞)、模型2组(M1型巨噬细胞与Ishikawa细胞、JAr细胞共培养)、温阳化浊方含药血清2组和空白血清2组。温阳化浊方含药血清2组和空白血清2组在M1型巨噬细胞与Ishikawa细胞共培养的同时,分别给予筛选浓度的温阳化浊方含药血清和空白血清干预48 h,再与JAr细胞共培养。Realtime PCR方法检测Wnt通路相关因子Wnt4、Wnt6、Wnt7b、Wnt11、β-连环蛋白(β-catenin)和子宫内膜容受性标志分子整合素αV (ITGαV)、整合素β3 (ITGβ3)、整合素β5 (ITGβ5)、E-钙黏蛋白(E-cadherin)、L-选择素(L-selectin)的mRNA表达;免疫荧光法检测Wnt通路下游分子原癌Objective To explore the possible mechanism of Wenyang Huazhuo Formula(温阳化浊方,WHF)in promoting embryo implantation.Methods Twenty rats were randomly divided into WHF group and blank group,with 10 rats in each group.Rats in the WHF group were given 21.05 g/kg of WHF liquid by gavage for 7 days,while those in the blank group was not given intragastric administration,so as to prepare WHF-containing serum and blank serum.THP-1 monocytes were cultured in vitro and were differentiated into macrophages induced by phorbol ester(PMA).Furthermore,20 ng·ml^(-1) lipopolysaccharide(LPS)and 20 ng·ml^(-1) gamma Interferon(IFN)-γ)were used to induce M1 polarization of macrophages.M1 macrophages were cultured in the 2%,4%,8%,10%,15%,and 20%WHF-containing serum and blank serum.CCK8 method was used to detect cell viability,and WHF-containing serum having the nontoxic maximum content were selected for follow-up experiment.M1 macrophages were divided into three groups:model 1 group,WHF-containing serum 1 group,and blank serum 1 group.After M1 macrophages polarization,WHF-containing serum 1 group was given selected concentration of WHF-containing serum,while blank serum 1 group was given same concentration of blank serum for 24 and 48 hours.Real-time fluorescence quantitative PCR(Real-time PCR)was used to detect mRNA expression of M1 macrophage markers including tumor necrosis factor-α(TNF-α)and interleukin 1β(IL-1β)and M2 macrophage markers including chemokine ligand 18(CCL-18)and mannose receptor type C-l(MRC-1).Transwell system was used to co-culture M1 macrophages,Ishikawa cells and JAr cells to establish an in vitro embryo implantation model.These cells were divided into five groups:non-receptive group(only Ishikawa cells),receptive group(including Ishikawa cells and JAr cells),model 2 group(M1 macrophages co-cultured with Ishikawa cells and JAr cells),WHF-containing serum 2 group and blank serum 2 group.While M1 macrophages were co-cultured with Ishikawa cells,the latter two groups were administered with selected

关 键 词：慢性子宫内膜炎 胚胎种植 温阳化浊方 子宫内膜容受性 巨噬细胞 极化 WNT通路 

分 类 号：R285.5[医药卫生—中药学]